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Retina  |   March 2010
Mutation- and Tissue-Specific Alterations of RPGR Transcripts
Author Affiliations & Notes
  • Fabian Schmid
    From the Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland;
  • Esther Glaus
    From the Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland;
  • Frans P. M. Cremers
    the Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; and
  • Barbara Kloeckener-Gruissem
    From the Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland;
    the Department of Biology, ETH Zurich, Switzerland.
  • Wolfgang Berger
    From the Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland;
  • John Neidhardt
    From the Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland;
  • Corresponding author: John Neidhardt, Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Schorenstrasse 16, 8603 Schwerzenbach, Switzerland; neidhardt@medgen.uzh.ch
Investigative Ophthalmology & Visual Science March 2010, Vol.51, 1628-1635. doi:10.1167/iovs.09-4031
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      Fabian Schmid, Esther Glaus, Frans P. M. Cremers, Barbara Kloeckener-Gruissem, Wolfgang Berger, John Neidhardt; Mutation- and Tissue-Specific Alterations of RPGR Transcripts. Invest. Ophthalmol. Vis. Sci. 2010;51(3):1628-1635. doi: 10.1167/iovs.09-4031.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The majority of patients with X chromosome-linked retinitis pigmentosa (XlRP) carry mutations in the RPGR gene. The authors studied whether patients with RPGR mutations show additional splice defects that may interfere with RPGR properties.

Methods.: Patient-derived cell lines with RPGR mutations were raised in suspension. To verify mutations, direct sequencing of PCR products was performed. Patient-specific alterations in RPGR splicing were analyzed by RT-PCR and confirmed by sequencing. Tissue-specific expression levels of RPGR splice variants were quantified by real-time PCR using pools of different human donor tissues.

Results.: The authors analyzed the splicing of RPGR in seven RP patient-derived lymphoblastoid cell lines carrying hemizygous RPGR mutations. In three patient cell lines, they identified and characterized splice defects that were present in addition to a mutation. These splice defects were likely to interfere with normal RPGR properties. Furthermore, they identified four novel RPGR transcripts, either containing a new exon termed 11a or skipping the constitutive exons 12, 14, and 15. Novel and known RPGR isoforms were found to be differentially regulated in several human tissues. In human retina, approximately 10% of RPGR transcripts are alternatively spliced between exons 9 and 15.

Conclusions.: These findings show that splicing of RPGR is precisely regulated in a tissue-dependent fashion and suggest that mutations in RPGR frequently interfere with the expression of alternative transcript isoforms. These results implicate the importance of RPGR transcript analysis in patients with RP. The authors further discuss RPGR splicing as a modifier of different disease phenotypes described in patients with XlRP.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous eye disorder leading to the degeneration of photoreceptors in the human retina. Most patients with RP exhibit difficulties in dark adaptation and night blindness in adolescence and loss of midperipheral vision in young adulthood. In general, the rod photoreceptors degenerate first, causing a loss of peripheral vision. During progression of the disease, cone photoreceptors may also die, which ultimately leads to loss of central vision and complete blindness. 1 The disease prevalence is approximately 1 in 3000 to 4000 people, affecting more than 1.5 million worldwide. More than 35 RP-associated genes have been identified so far. Most of them have been reported to be involved in phototransduction, basic metabolic pathways, maintenance of photoreceptor morphology, transcriptional regulation, or pre-mRNA splicing, as reviewed by Kennan et al. 2  
The inheritance pattern of RP can be autosomal recessive (arRP), autosomal dominant (adRP), or X-linked (XlRP). The latter is considered to cause the clinically severest form of RP. Approximately 60% to 70% of all X-linked families show mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. 3,4 Here, the majority of mutations were found in the alternatively spliced exon ORF15, whereas exons 1 to 15 carry a minor portion of all RPGR mutations. 59 A few mutations in exon 1 to 15 of RPGR have been associated with additional nonocular manifestations. In a family with XlRP described previously, two members displayed a complex phenotype combining primary ciliary dyskinesia and retinitis pigmentosa. In this case, aberrant splicing of RPGR was found in nasal epithelial cells because of a 57-bp deletion in exon 6. 10 Additionally, a syndromic phenotype including RP, hearing defects, and severe sinorespiratory infections has been associated with mutations in RPGR exon 6 4 or exon 8. 11 These reports indicate that the function of the widely expressed RPGR is important not only for the retina but also for other tissues. 
RPGR transcripts have been identified in several human tissues (e.g., retina, brain, lung, kidney, and testis). 1214 Moreover, transcript isoforms of RPGR have been described and occasionally found to be expressed in a tissue- or cell-type-specific manner. One of these transcripts includes the alternative exon 15a, 14 and another contains exon 15b. 15 RPGR isoforms may also show skipping of constitutive exons (e.g., exons 14 and 15). 14 Transcripts containing the alternative exon 9a produce a truncated protein that is predominantly expressed in human cone photoreceptors. 16  
The effect of many RPGR mutations on protein structure has been predicted. 5,6,1215,17 However, as has been described for other genes, a large fraction of exonic mutations also leads to splice defects by disruption of constitutive and regulatory splice-relevant sequences. 18 Because mutation-induced alterations of splice patterns in RPGR may contribute to the pathogenic mechanism for RP, 16,1922 we analyzed the effect of different RPGR mutations on splicing. Four novel RPGR isoforms were identified that showed differential expression in several tissues. Our results suggest that RPGR splicing is part of the pathogenic mechanism underlying the complex disease of RP. This further raises the possibility that alterations in RPGR splicing act as modifiers of the disease expression. 
Materials and Methods
Cell Culture
Splicing of RPGR was analyzed in seven Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) derived from the blood of male patients with RP (Table 1) and an unaffected male control. Informed consent was obtained from each patient and the unaffected control in this study, and all were treated in accordance with the Declaration of Helsinki. Additional family members were not available for further tests. LCLs were grown in RPMI 1640 medium (LabForce, Nunningen, Switzerland), 10% FBS (LabForce), 1.1% penicillin-streptomycin (LabForce), and 1.3% L-glutamine (LabForce) at 37°C, 5% CO2 by using 75-cm2 cell culture flasks (Techno Plastic Products, Trasadingen, Switzerland). Between 8 × 106 and 4 × 107 cells were pelleted by centrifugation at 4°C with 2000g for 10 minutes. The pellets were washed with 1× DPBS (LabForce), snap frozen, and stored at −80°C before extraction of DNA or RNA. To inhibit nonsense-mediated mRNA decay (NMD), 1 × 106 cells/mL were incubated in 30 μg/mL cycloheximide (Sigma-Aldrich, Schnelldorf, Germany) at 37°C, 5% CO2 in 25-cm2 cell culture flasks (Techno Plastic Products) for 4 hours, as previously described. 23  
Table 1.
 
RPGR Mutations of Analyzed Lymphoblastoid Cell Lines Derived from Male Patients with X1RP
Table 1.
 
RPGR Mutations of Analyzed Lymphoblastoid Cell Lines Derived from Male Patients with X1RP
Patient ID Mutation in Exon/Intron Sequence Alteration Consequence of Sequence Alteration
2616 Intron 1 c.28+1G>A Potential splice defect
4767* Exon 5 c.389T>G p.Phe130Cys
Exon 11a g.31259G>A† ‡ Splice defect/p.Asp484Asn
2884 Exon 7 c.706C>T p.Glu236X
2555 Exon 8 c.823G>A p.Gly275Ser
2603 Exon 8 c.823G>A p.Gly275Ser
2549 Exon 11 c.1399C>T p.Gln467X
2550§ Exon 11 c.1402_1405delCCAG p.Arg468fs
RNA Extraction, cDNA Synthesis, and RT-PCR
To extract total RNA from LCLs, the cells were disrupted using either spin columns (QIAshredder; Qiagen, Hombrechtikon, Switzerland) or a rotor-stator homogenizer (Ultra-Turrax T8; IKA Analysetechnik GmbH, Heitersheim, Germany). Subsequently, RNA was isolated with kits (RNeasy Mini or Midi; Qiagen) according to the manufacturer's instructions. RNA quality was verified with an electrophoresis analysis system (2100 Bioanalyzer; Agilent, Basel, Switzerland), and the amount was determined with a photometer (ND-1000; NanoDrop, Litau, Switzerland). One thousand nanograms of total RNA were randomly primed before reverse transcription by reverse transcriptase (Superscript III; Invitrogen, Basel, Switzerland) according to the manufacturer's protocol. To verify correct splicing of the RPGR transcripts, RT-PCR reactions were performed with 50 ng cDNA, as described previously. 16 Primers used in the RT-PCR assays are shown in Supplementary Table S1. RT-PCR products were confirmed by sequencing. Each RT-PCR experiment was replicated three times using RNA from independently grown LCL cells. 
Quantitative Real-Time PCR of Alternative RPGR Transcript Variants
Quantifications of RPGR transcript levels were performed by quantitative real-time PCR (HT 7900 TaqMan using Assay by Design MGB FAM-TAMRA-labeled probes; Applied Biosystems, Rotkreuz, Switzerland). To study the expression of RPGR transcript isoforms in several tissues, total RNA from pools of different human donor tissues (brain, n = 5; kidney, n = 5; lung, n = 3; retina, n = 25; testis, n = 39) was purchased from Becton Dickinson (Allschwil, Switzerland) or BioCat (Heidelberg, Germany). Three to four cDNA batches were independently generated and analyzed from each tissue. 
We designed probes (TaqMan; Applied Biosystems) to quantify exon/exon boundaries for each alternative RPGR transcript isoform and a corresponding probe for the constitutive transcript. Probes specific to the constitutive transcript of RPGR were used as endogenous controls. Their comparability was tested according to the manufacturer's instructions. Each quantification reaction was technically replicated at least four times and gave results with amplification curves starting between 25 and 30 cycles. Using equal amounts of cDNA per sample, Ct values of the constitutive transcript did not differ significantly between tissues (errors represent 95% confidence intervals [CIs]): retina 26.3 (±0.86), brain 25.75 (±1.45), lung 25.9 (±0.37), kidney 26.53 (±1.23), and testis 25.6 (±1.49). The outcome was analyzed using specialized software (SDS 2.2; Applied Biosystems). Expression levels of the tested tissues or LCLs were normalized to human retina or an unaffected male control, respectively; 95% CIs were calculated from three to four independently generated samples. 
DNA Purification and Sequencing
RT-PCR products were gel-extracted (QIAquick Gel Extraction Kit; Qiagen). Gel-purified DNA was either cloned into the vector (pCRII-TOPO; Topo TA Cloning Kit Dual Promoter; Invitrogen) according to the manufacturer's instructions or sequenced directly. DNA from LCLs was extracted as previously described. 24 To identify or verify mutations in patient cell lines, exons of RPGR were amplified by PCR (HotFirePol; Solis Biodyne, Tartu, Estonia) and sequenced directly (sequencing analyzer, model 3100; Applied Biosystems). Mutations were designated according to the nomenclature provided by the human genome variation society (www.hgvs.org). The numbering of mutations refers to the human RPGR reference sequence NM_001034853 for cDNA or reference assembly NC_000023.9 (range, 38013367–38071732) for genomic DNA from the National Center for Biotechnology Information database. The potential effect of exonic mutations on exonic splicing enhancer (ESE) binding sites was analyzed (ESEfinder 3.0; http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process = home). 
Results
We followed the hypothesis that splice defects frequently occur in addition to mutations found in RPGR. To detect splice defects, we established RT-PCR assays that allow analysis of RPGR transcripts and screened LCLs derived from seven male patients with RP affected by known hemizygous RPGR mutations (Table 1). The LCLs used were not preselected for possible splice defects. 
Splice Site Mutation in RPGR Intron 1
In patient cell line LCL 2616, RT-PCR amplification of exons 1 through 5 yielded no product (Fig. 1). In contrast, using primers that bind to exons 2 and 5 resulted in a fragment, although weaker than that of the unaffected control and other cell lines. Furthermore, RT-PCR with primer combinations specific to downstream located exons confirmed that LCL 2616 shows reduced RPGR transcript amounts (Fig. 1). These analyses suggested the presence of a mutation affecting normal transcript processing in this patient cell line. Indeed, a mutation at the first base of intron 1 (c.28+1G>A; Table 1) was present in LCL 2616. This mutation is expected to result in a splice defect. Although extensively assayed, we were not able to amplify splice products between exon 1 and 2. These results suggest defective splicing of RPGR because of the mutation c.28+1G>A leading to a reduced amount of transcripts. It further raises the possibility that the translation of RPGR may start from exon 3, where the next in-frame start codon is located. 
Figure 1.
 
Characterization of a splice site mutation in LCL 2616. RPGR transcript analysis in the patient cell line 2616 and the unaffected control using different primer combinations that span the whole RPGR transcript. DNA marker sizes are given in base pairs.
Figure 1.
 
Characterization of a splice site mutation in LCL 2616. RPGR transcript analysis in the patient cell line 2616 and the unaffected control using different primer combinations that span the whole RPGR transcript. DNA marker sizes are given in base pairs.
The Novel RPGR Exon 11a
We performed RT-PCR analyses of RPGR transcripts containing exons 11 through 15 in all patient cell lines listed in Table 1. In LCL 4767 we detected an additional amplicon of approximately 700 bp in length by conventional RT-PCR (Fig. 2A). Sequencing revealed that this fragment includes a novel exon spliced between exons 11 and 12. It was thus termed exon 11a. It contains 160 nucleotides and starts 1139 bp downstream of the splice donor site of exon 11 (Fig. 2B). The resultant new coding sequence contains a stop codon. 
Figure 2.
 
Overexpression of the RPGR +Ex11a transcript isoform in patient cell line 4767. (A) RT-PCR amplification of RPGR transcripts containing exons 11 through 15. Schematic drawings illustrate the exon composition of amplified RT-PCR products. Primer binding sites are indicated as horizontal gray bars. DNA marker sizes are given in base pairs. (B) Sequence of the novel RPGR exon 11a and flanking intronic regions. Nucleotides in gray represent exon 11a sequences, whereas deduced amino acids are indicated as single letter code above of each nucleotide triplet. The stop codon is presented in italics, and coding sequences are shown in uppercase letters. The position mutated in patient 4767 (g.31259G>A) is marked by a box. NC_000023.9 was used as reference sequence for genomic DNA (numbers at the right indicate the nucleotide position). (C) Real-time quantitative RT-PCR of exon 11a expression levels detected in the unaffected control (I), in a pool of six LCLs carrying hemizygous RPGR mutations (n = 6) different from the sequence alteration in exon 11a (II) and in the patient cell line 4767 (III). Error bars represent 95% CI calculated from cDNA batches generated from the RNA of three independently grown LCL cells. Asterisk indicates statistical significance between measurements. (D) Electropherograms from parts of the exon 11a sequence of patient cell line 4767 and a control. Arrows indicate the position of the mutation g.31259G>A in patient 4767. The stop codon is shown in italics. Positions of putative binding sites for ESEs SF2/ASF and SC35 are depicted as horizontal bars. The results suggest the existence of an additional SC35 binding site uniquely in the patient.
Figure 2.
 
Overexpression of the RPGR +Ex11a transcript isoform in patient cell line 4767. (A) RT-PCR amplification of RPGR transcripts containing exons 11 through 15. Schematic drawings illustrate the exon composition of amplified RT-PCR products. Primer binding sites are indicated as horizontal gray bars. DNA marker sizes are given in base pairs. (B) Sequence of the novel RPGR exon 11a and flanking intronic regions. Nucleotides in gray represent exon 11a sequences, whereas deduced amino acids are indicated as single letter code above of each nucleotide triplet. The stop codon is presented in italics, and coding sequences are shown in uppercase letters. The position mutated in patient 4767 (g.31259G>A) is marked by a box. NC_000023.9 was used as reference sequence for genomic DNA (numbers at the right indicate the nucleotide position). (C) Real-time quantitative RT-PCR of exon 11a expression levels detected in the unaffected control (I), in a pool of six LCLs carrying hemizygous RPGR mutations (n = 6) different from the sequence alteration in exon 11a (II) and in the patient cell line 4767 (III). Error bars represent 95% CI calculated from cDNA batches generated from the RNA of three independently grown LCL cells. Asterisk indicates statistical significance between measurements. (D) Electropherograms from parts of the exon 11a sequence of patient cell line 4767 and a control. Arrows indicate the position of the mutation g.31259G>A in patient 4767. The stop codon is shown in italics. Positions of putative binding sites for ESEs SF2/ASF and SC35 are depicted as horizontal bars. The results suggest the existence of an additional SC35 binding site uniquely in the patient.
These results suggested elevated expression levels of RPGR transcripts containing exon 11a (RPGR + Ex11a) in LCL 4767. We confirmed this observation by quantitative RT-PCR and detected a fourfold increased expression of RPGR + Ex11a only in this patient cell line (Fig. 2C). These results document a splice alteration specific to LCL 4767. 
The previously identified missense mutation in exon 5 (c.289T>G, p.Phe130Cys; Table 1) in this patient 13 is most likely not causing the overexpression of exon 11a because of the distance between the two affected exons. Therefore, we searched for additional sequence alterations in exon 11a and identified a G>A transition at position 36 (g.31259G>A), 15 bp upstream of the stop codon of exon 11a (Fig. 2D, Table 1). The sequence alteration was not annotated as a single nucleotide polymorphism (SNP) in databases (UCSC, ENSEMBL, NCBI) and was not found in 300 ethnically matched control alleles (Caucasians), suggesting a pathogenic nature of this variant. 
Application of the ESEfinder program to this sequence alteration predicted a novel binding site for the splice factor SC35 only in the mutated exon 11a (Fig. 2D). Enhanced binding of SC35 to exon 11a may thus explain the significantly elevated levels of RPGR + Ex11a transcripts specifically found in patient 4767. 
RPGR Exon 12, 14, or 15 Skipping
RT-PCR analysis of RPGR transcripts, including exons 11 through 19, revealed two major isoforms either containing all known exons or simultaneously skipping exons 14 and 15 (Fig. 3A). The control samples showed an additional band at 1000 bp (Fig. 3A). Cloning and sequencing of this band documented that this amplicon is a mixture of two thus far unknown RPGR isoforms that skip either exon 14 (RPGR skipEx14) or 15 (RPGR skipEx15). 
Figure 3.
 
Upregulation of the novel RPGR skipEx12 transcript. (A) RT-PCR amplification of exons 11 to 19 of the RPGR transcript. Schematic drawings illustrate the sequence of amplified RT-PCR products. Each RT-PCR product is marked by a number corresponding to the respective schematic drawing. Primer binding sites in exons 11 and 19 are indicated by horizontal gray bars. Numbers at the left represent DNA marker sizes and are given in base pairs. (B) RT-PCR of RPGR exons 11 to 19 of patient cell line 2550 and a control, either incubated with (+) or without (−) CHX to inhibit NMD. The RT-PCR reaction lacking a template is indicated as H2O. (C) Graphical illustration of mutation-induced mechanisms in patient cell line 2550 leading to detectable levels of RPGR skipEx12 transcripts. The asterisk labels a premature termination codon (PTC) in exon 12, which is generated in patient cell line 2550 because of the 4-bp deletion in exon 11. The PTC causes reduced expression levels of transcripts, including exon 12, by NMD (upper). In contrast, transcripts lacking exon 12 in combination with the 4-bp deletion escape NMD because of preservation of the open reading frame (lower).
Figure 3.
 
Upregulation of the novel RPGR skipEx12 transcript. (A) RT-PCR amplification of exons 11 to 19 of the RPGR transcript. Schematic drawings illustrate the sequence of amplified RT-PCR products. Each RT-PCR product is marked by a number corresponding to the respective schematic drawing. Primer binding sites in exons 11 and 19 are indicated by horizontal gray bars. Numbers at the left represent DNA marker sizes and are given in base pairs. (B) RT-PCR of RPGR exons 11 to 19 of patient cell line 2550 and a control, either incubated with (+) or without (−) CHX to inhibit NMD. The RT-PCR reaction lacking a template is indicated as H2O. (C) Graphical illustration of mutation-induced mechanisms in patient cell line 2550 leading to detectable levels of RPGR skipEx12 transcripts. The asterisk labels a premature termination codon (PTC) in exon 12, which is generated in patient cell line 2550 because of the 4-bp deletion in exon 11. The PTC causes reduced expression levels of transcripts, including exon 12, by NMD (upper). In contrast, transcripts lacking exon 12 in combination with the 4-bp deletion escape NMD because of preservation of the open reading frame (lower).
Comparing all patient-derived cell lines, LCL 2550 showed an alteration from the control splice pattern in conventional RT-PCR analyses. We detected two additional fragments, both migrating 92 bp below the two major products. As confirmed by sequencing, these novel transcripts lack exon 12 of RPGR (RPGR skipEx12; Fig. 3A). 
Patient 2550 carries a deletion of 4 bp in exon 11 (c.1402_1405delCCAG, p.Arg468fs; Table 1). 13 This deletion affects positions −12 to −9 upstream of the last nucleotide of exon 11, an exonic region spatially unrelated to the splice donor site. Importantly, the deletion causes a frame-shift that leads to a premature termination codon (PTC) in exon 12 (Fig. 3C). Because transcripts containing a PTC are susceptible to NMD, 25 we searched for such potential effects. To inhibit NMD, we treated cells with cycloheximide (CHX) and compared them with untreated cells. We found only in LCL 2550 that inhibition of NMD affects splicing and restores the amplification pattern identified in the control cell line (Fig. 3B). These findings show that the 4-bp deletion in exon 11 causes a PTC in exon 12 that activates partial degradation by NMD. 
Interestingly, LCL 2550 showed a transcript combining exon 12 skipping (deletion of 92 bp) and deleting 4 bp in exon 11. This combination restores the open reading frame in the RPGR transcript (Fig. 3C). Thus, a protein might be translated that lacks amino acids coded by exon 12. 
Tissue-Specific Expression Analysis of RPGR Isoforms
Our studies identified the four novel RPGR transcripts RPGR +Ex11a, RPGR skipEx12, RPGR skipEx14, and RPGR skipEx15. To verify whether they are also generated in human tissues other than lymphoblastoid cell lines, transcript analysis was performed by quantitative RT-PCR using cDNA from pools of different human tissues, including retina, brain, lung, kidney, and testis. In addition, we tested whether the expression of previously reported RPGR isoforms also shows tissue-specific variation. This included RPGR +Ex9a, which has recently been found to be expressed predominantly in cone photoreceptors of the human retina 16 and transcripts, simultaneously skipping exons 14 and 15 (RPGR skipEx14/15). 14 The assessment of isoform quantities was performed in comparison to the constitutive transcript of RPGR
Our data show that the abundance of each of the alternative RPGR isoforms varied between 0.8% (RPGR skipEx12) and 2.7% (RPGR skipEx14/15) within the retina (Fig. 4). In summary, we document that in the human retina, approximately 10% of RPGR transcripts are alternatively spliced between exons 9 and 15 (Fig. 4). 
Figure 4.
 
Relative abundance of alternative transcript isoforms of RPGR in human retina. Expression levels of alternative variants are displayed as percentages of the constitutive RPGR transcript in human retina. Numbers in bars represent mean values given in percentages Error bars represent confidence intervals of 95% CIs calculated from three to four independently generated cDNA batches.
Figure 4.
 
Relative abundance of alternative transcript isoforms of RPGR in human retina. Expression levels of alternative variants are displayed as percentages of the constitutive RPGR transcript in human retina. Numbers in bars represent mean values given in percentages Error bars represent confidence intervals of 95% CIs calculated from three to four independently generated cDNA batches.
The amount of each alternative isoform also varied among different tissues (Fig. 5). The highest levels of RPGR +Ex11a were observed in retina, whereas other tissues contained significantly lower amounts (Fig. 5A). Furthermore, we detected the highest expression of RPGR skipEx12 in lung and kidney and found 10- to 12-fold lower amounts of this isoform in retina, brain, and testis (Fig. 5B). Similarly, RPGR + Ex9a transcripts were expressed more greatly in lung and kidney than in retina (Fig. 5E). The highest expression of the novel RPGR variant RPGR skipEx14 was found in brain (Fig. 5C), whereas RPGR skipEx15 expression was lowest in retina (Fig. 5D). Isoform RPGR skipEx14/15 exhibited the largest variation: it was 20 times more highly expressed in brain than in retina (Fig. 5F). 
Figure 5.
 
Multi-tissue expression analyses of novel and previously reported RPGR transcript isoforms. Quantitative RT-PCR to detect expression of (A) RPGR +Ex11a, (B) RPGR skipEx12, (C) RPGR skipEx14, (D) RPGR skipEx15, (E) RPGR +Ex9a, and (F) RPGR skipEx14/15 from different donor tissues. For quantification of the novel RPGR transcript isoform, we used expression of the constitutive RPGR transcript as endogenous control. Subsequently, relative expression levels of each isoform were compared with human retina (retinal levels were set to 1). Asterisks illustrate significance between measurements as described in the Results section. Error bars represent 95% CIs calculated from three to four independently generated cDNA batches.
Figure 5.
 
Multi-tissue expression analyses of novel and previously reported RPGR transcript isoforms. Quantitative RT-PCR to detect expression of (A) RPGR +Ex11a, (B) RPGR skipEx12, (C) RPGR skipEx14, (D) RPGR skipEx15, (E) RPGR +Ex9a, and (F) RPGR skipEx14/15 from different donor tissues. For quantification of the novel RPGR transcript isoform, we used expression of the constitutive RPGR transcript as endogenous control. Subsequently, relative expression levels of each isoform were compared with human retina (retinal levels were set to 1). Asterisks illustrate significance between measurements as described in the Results section. Error bars represent 95% CIs calculated from three to four independently generated cDNA batches.
Taken together, these findings show that the expression of alternative RPGR transcripts varies significantly among and within tissues. This indicates distinct tissue-specific RPGR splicing that may confer specialized function of the different isoforms. 
Discussion
Alternative splicing of pre-mRNA transcripts is considered to be an important mechanism to increase protein variability from a single gene. 26 This process also acts on human RPGR and generates alternative transcript isoforms, the expression of which is spatially and temporally regulated. 1416  
In this article, we describe four novel transcript isoforms of RPGR termed RPGR + Ex11a, RPGR skipEx12, RPGR skipEx14, and RPGR skipEx15. We showed that all are expressed in a tissue-dependent fashion, which suggests functional consequences. The highest expression levels of both RPGR skipEx14 and RPGR skipEx14/15 were detected in brain. Consequently, misregulation of transcripts lacking exon 14 or exons 14 and 15 might disturb their function in brain. To date, no mutation in RPGR has been described that shows a phenotypic manifestation in this tissue. However, only a few patients are known to contain mutations in exon 14 or 15 (compare to http://rpgr.hgu.mrc.ac.uk). Detailed clinical characterization of the brain functions in these patients might add valuable information on the phenotypic spectrum caused by mutations in RPGR
Our data suggest that RPGR transcripts are differentially spliced among tissues. They further show that the novel isoforms described herein are expressed at lower levels than the constitutive transcript of RPGR. Nevertheless, a significant portion of RPGR transcripts are alternatively spliced between exons 9 and 15 in the retina. Because tissues are heterogenous with respect to cell type, one possible explanation for our observation is that only a fraction of cells within a given tissue expresses a distinct isoform or that expression is cell type specific. Similar observations have been published for RPGR containing exon 9a. 16 The corresponding protein of the exon 9a-containing transcript was predominantly present in cones of the retina, suggesting a specialized function of this isoforms in a subfraction of photoreceptors. It is a matter of future study to evaluate whether the novel RPGR isoforms described herein are also expressed by a specific cell type within distinct tissues. 
Regulation of alternative and tissue-specific splicing is thought to be controlled by proteins that stimulate or repress exon recognition. In general, such factors are called exonic or intronic splicing enhancers (ESEs or ISEs) and silencers. Some of these factors are expressed only in certain tissues and regulate splicing of specific sets of pre-mRNAs. 27 A well-known example is the mammalian splicing factor NOVA-1, which is involved in the control of alternative pre-mRNA splicing in neurons. This protein binds specific intronic sequences of pre-mRNA targets through its K-homology (KH) domains. NOVA-1 is reported to enhance the inclusion of specific alternative exons into target transcripts and is expressed in distinct populations of neurons in the brain. 28 Taking this into account, we envision that factors with similar functions might regulate the expression of alternative RPGR transcript isoforms within tissues. 
A different mechanism of gene regulation acts through NMD, which constitutes a posttranscriptional regulatory process. We found that NMD reduces the amount of a mutated RPGR transcript that contains a PTC. Of note, NMD may also act on nonmutated transcripts. 2931 In addition, it has been documented that it can be active in a tissue-specific manner. 3234 These observations provide a possible explanation why RPGR isoforms were found to be expressed at elevated levels among tissues. 
Interestingly, most RPGR isoforms truncate the deduced protein downstream of the regulator of chromosome condensation 1-like domain (RCC1). The RCC1-like domain of RPGR includes exons 2 through 11. 5,14,16 This domain has been shown to bind four different interaction partners: RPGR interacting protein 1 (RPGRIP1), prenyl-binding protein delta, and structural maintenance of chromosomes 1 and 3 proteins. 3538 Different RPGR protein isoforms have been shown to be associated with a multiprotein complex, which is thought to be involved in intraphotoreceptor protein transport through the connecting cilium. 39 We have previously shown that exon 9a containing RPGR transcripts gives rise to a truncated protein that binds distinct isoforms of RPGRIP1. 16 On the assumption that the other transcript variants described herein are also translated, our data support a model that predicts binding of distinct RPGR isoforms to different sets of interaction partners to form various functionally unique protein complexes. Mutations that modify distinct RPGR isoforms would influence the formation of these complexes. Cases have been reported in which RPGR mutations in the RCC1-like domain (affecting exons 6 and 8) are associated with nonocular manifestations. 4,10,11 These reports further support our hypothesis that RPGR isoform-specific protein complexes fulfill tissue-specific functions. 
The disease phenotype in patients affected by RPGR mutations can be highly variable even within one family. These observations imply the existence of modifiers that influence the severity of the disease. Here we showed that not only does a mutation in RPGR affect the predicted amino acid sequence of the protein, it may also result in missplicing or altered regulation of specific transcripts. Given that alternative splicing is precisely regulated in a tissue-specific and a developmental stage-dependent manner, changes in this process may cause or modify disease symptoms. 40 Taking this into account, our results suggest that defects in RPGR splicing could modify the disease phenotype of RP. Several examples for splicing-induced modifications of a disease have been reported. 41  
We have identified alterations in the splicing of RPGR in 3 of 7 cell lines of patients with RP. Our findings implicate the importance of including the analysis of RPGR splicing in patients with retinal degenerations and associated phenotypes. Furthermore, patient-derived cell lines showed splice alterations in RPGR isoforms that are differentially regulated among distinct human tissues. Thus, not only could a change in the expression of these isoforms affect the retina, it could also disrupt their function in other tissues. 
In patient cell line 2550, we detected elevated levels of RPGR skipEx12 transcripts. The deletion of 4 bp in combination with exon 12 skipping abolishes the deleterious effects of the frame-shift. Therefore, these transcripts can now escape degradation by NMD and may be expressed as a truncated RPGR protein in the patient. The splice defect is likely to take place in most tissues, indicating that more than the retina is affected. Interestingly, there is evidence that patient 2550 has a mild hearing impairment. The audiogram showed a dip with high-frequency pure tones (Rosenberg T, personal communication, 2007–2008). Although speculatory, the nonocular manifestation of auditory problems might be the result of the expression of this aberrant protein isoform. It has been observed in a different patient (patient ID 2557) that a 6.4-kb deletion within RPGR, 17 in addition to a 1-bp deletion at the 3′ end of exon ORF15 (c.3395delA), 6 may affect the audiogram. 42 Nevertheless, an age-related effect on hearing in these patients cannot be excluded. It would further be of interest to clarify whether the respiratory function of patient 2550 is impaired because our expression data suggest that RPGR skipEx12 transcripts might be relevant in lung. 
We showed that patient 4767 expresses increased levels of RPGR +Ex11a, likely because of a sequence alteration in exon 11a. This novel mutation is predicted to change the activity of a splice enhancer site of SC35, which might control the frequency of exon 11a inclusion into the transcript. SC35, a ubiquitously expressed splice factor, has been shown to increase the integration of a target exon by mediating specific interactions with components of the spliceosome. 43 Mutations that increase the binding affinity for an exonic splicing enhancer have already been associated with other human diseases, e.g., frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17) 44 and a cardiac phenotype of Fabry disease. 45 We demonstrated that the expression of exon 11a was significantly increased only in cell line 4767. Toxic effects leading to premature death of photoreceptors might be caused by overexpression of a distinct RPGR isoform, a mechanism similar to that proposed by us for RPGR +Ex9a. 16 Furthermore, the overexpression of RPGR in mice also leads to a severe phenotype in sperm flagella, resulting in male infertility. 46  
Interestingly, the family pedigree of patient 4767 shows over a period of six generations an inheritance pattern by which female carriers are also affected (data not shown). We cannot exclude that this mode of inheritance is caused by skewed X-inactivation in affected females. However, this mechanism has never been found to influence the phenotype of female carriers of RPGR mutations 6,47,48 and, thus, is unlikely to be a common mechanism explaining symptomatic female carriers. In the patient described herein, either a cumulative or a separate effect of the two identified sequence alternation in exons 5 and 11a might be the cause of the disease. 
Taken together, the results reported here provide new insights into alternative splicing of RPGR and will help to elucidate its role in distinct tissues. Moreover, RPGR splice defects provide a pathogenic mechanism newly associated with RPGR, which implies that classical and syndromic patients with RP should be analyzed for alterations in RPGR splicing. 
Supplementary Materials
Footnotes
 Supported by the Velux Foundation, the Olga Mayenfisch Foundation, and Forschungskredit der Universität Zürich (JN).
Footnotes
 Disclosure: F. Schmid, None; E. Glaus, None; F.P.M. Cremers, None; B. Kloeckener-Gruissem, None; W. Berger, None; J. Neidhardt, None
The authors thank Silke Feil for cell culture support, and Christina Reinhard and Heymut Omran for providing a control cell line. 
References
Hartong DT Berson EL Dryja TP . Retinitis pigmentosa. Lancet. 2006;368:1795–1809. [CrossRef] [PubMed]
Kennan A Aherne A Humphries P . Light in retinitis pigmentosa. Trends Genet. 2005;21:103–110. [CrossRef] [PubMed]
Breuer DK Yashar BM Filippova E . A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa. Am J Hum Genet. 2002;70:1545–1554. [CrossRef] [PubMed]
Iannaccone A Breuer DK Wang XF . Clinical and immunohistochemical evidence for an X linked retinitis pigmentosa syndrome with recurrent infections and hearing loss in association with an RPGR mutation. J Med Genet. 2003;40:e118. [CrossRef] [PubMed]
Vervoort R Wright AF . Mutations of RPGR in X-linked retinitis pigmentosa (RP3). Hum Mutat. 2002;19:486–500. [CrossRef] [PubMed]
Neidhardt J Glaus E Lorenz B . Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing. Mol Vis. 2008;14:1081–1093. [PubMed]
Pelletier V Jambou M Delphin N . Comprehensive survey of mutations in RP2 and RPGR in patients affected with distinct retinal dystrophies: genotype-phenotype correlations and impact on genetic counseling. Hum Mutat. 2007;28:81–91. [CrossRef] [PubMed]
Sharon D Sandberg MA Rabe VW Stillberger M Dryja TP Berson EL . RP2 and RPGR mutations and clinical correlations in patients with X-linked retinitis pigmentosa. Am J Hum Genet. 2003;73:1131–1146. [CrossRef] [PubMed]
Shu X McDowall E Brown AF Wright AF . The human retinitis pigmentosa GTPase regulator gene variant database. Hum Mutat. 2008;29:605–608. [CrossRef] [PubMed]
Moore A Escudier E Roger G . RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa. J Med Genet. 2006;43:326–333. [CrossRef] [PubMed]
Zito I Downes SM Patel RJ . RPGR mutation associated with retinitis pigmentosa, impaired hearing, and sinorespiratory infections. J Med Genet. 2003;40:609–615. [CrossRef] [PubMed]
Meindl A Dry K Herrmann K . A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3). Nat Genet. 1996;13:35–42. [CrossRef] [PubMed]
Roepman R van Duijnhoven G Rosenberg T . Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1. Hum Mol Genet. 1996;5:1035–1041. [CrossRef] [PubMed]
Kirschner R Rosenberg T Schultz-Heienbrok R . RPGR transcription studies in mouse and human tissues reveal a retina-specific isoform that is disrupted in a patient with X-linked retinitis pigmentosa. Hum Mol Genet. 1999;8:1571–1578. [CrossRef] [PubMed]
Vervoort R Lennon A Bird AC . Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa. Nat Genet. 2000;25:462–466. [CrossRef] [PubMed]
Neidhardt J Glaus E Barthelmes D Zeitz C Fleischhauer J Berger W . Identification and characterization of a novel RPGR isoform in human retina. Hum Mutat. 2007;28:797–807. [CrossRef] [PubMed]
Roepman R Bauer D Rosenberg T . Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XlRP). Hum Mol Genet. 1996;5:827–833. [CrossRef] [PubMed]
Wang GS Cooper TA . Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet. 2007;8:749–761. [CrossRef] [PubMed]
Fujita R Buraczynska M Gieser L . Analysis of the RPGR gene in 11 pedigrees with the retinitis pigmentosa type 3 genotype: paucity of mutations in the coding region but splice defects in two families. Am J Hum Genet. 1997;61:571–580. [CrossRef] [PubMed]
Bauer S Fujita R Buraczynska M . Phenotype of an X-linked retinitis pigmentosa family with a novel splice defect in the RPGR gene. Invest Ophthalmol Vis Sci. 1998;39:2470–2474. [PubMed]
Dry KL Manson FD Lennon A Bergen AA Van Dorp DB Wright AF . Identification of a 5′ splice site mutation in the RPGR gene in a family with X-linked retinitis pigmentosa (RP3). Hum Mutat. 1999;13:141–145. [CrossRef] [PubMed]
Demirci FY Radak AL Rigatti BW Mah TS Gorin MB . A presumed missense mutation of RPGR causes abnormal RNA splicing with exon skipping. Am J Ophthalmol. 2004;138:504–505. [CrossRef] [PubMed]
Chatr-Aryamontri A Angelini M Garelli E . Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia. Hum Mutat. 2004;24:526–533. [CrossRef] [PubMed]
Neidhardt J Barthelmes D Farahmand F Fleischhauer JC Berger W . Different amino acid substitutions at the same position in rhodopsin lead to distinct phenotypes. Invest Ophthalmol Vis Sci. 2006;47:1630–1635. [CrossRef] [PubMed]
Maquat LE . When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995;1:453–465. [PubMed]
Black DL . Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell. 2000;103:367–370. [CrossRef] [PubMed]
Maniatis T Tasic B . Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature. 2002;418:236–243. [CrossRef] [PubMed]
Dredge BK Polydorides AD Darnell RB . The splice of life: alternative splicing and neurological disease. Nat Rev Neurosci. 2001;2:43–50. [CrossRef] [PubMed]
Rehwinkel J Letunic I Raes J Bork P Izaurralde E . Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets. RNA. 2005;11:1530–1544. [CrossRef] [PubMed]
Wittmann J Hol EM Jack HM . hUPF2 silencing identifies physiologic substrates of mammalian nonsense-mediated mRNA decay. Mol Cell Biol. 2006;26:1272–1287. [CrossRef] [PubMed]
Mendell JT Sharifi NA Meyers JL Martinez-Murillo F Dietz HC . Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat Genet. 2004;36:1073–1078. [CrossRef] [PubMed]
Linde L Boelz S Neu-Yilik G Kulozik AE Kerem B . The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells. Eur J Hum Genet. 2007;15:1156–1162. [CrossRef] [PubMed]
Weischenfeldt J Damgaard I Bryder D . NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements. Genes Dev. 2008;22:1381–1396. [CrossRef] [PubMed]
Bateman JF Freddi S Nattrass G Savarirayan R . Tissue-specific RNA surveillance? Nonsense-mediated mRNA decay causes collagen X haploinsufficiency in Schmid metaphyseal chondrodysplasia cartilage. Hum Mol Genet. 2003;12:217–225. [CrossRef] [PubMed]
Linari M Ueffing M Manson F Wright A Meitinger T Becker J . The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase. Proc Natl Acad Sci U S A. 1999;96:1315–1320. [CrossRef] [PubMed]
Boylan JP Wright AF . Identification of a novel protein interacting with RPGR. Hum Mol Genet. 2000;9:2085–2093. [CrossRef] [PubMed]
Roepman R Bernoud-Hubac N Schick DE . The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors. Hum Mol Genet. 2000;9:2095–2105. [CrossRef] [PubMed]
Khanna H Hurd TW Lillo C . RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins. J Biol Chem. 2005;280:33580–33587. [CrossRef] [PubMed]
He S Parapuram SK Hurd TW . Retinitis pigmentosa GTPase regulator (RPGR) protein isoforms in mammalian retina: insights into X-linked retinitis pigmentosa and associated ciliopathies. Vision Res. 2008;48:366–376. [CrossRef] [PubMed]
Nissim-Rafinia M Kerem B . Splicing regulation as a potential genetic modifier. Trends Genet. 2002;18:123–127. [CrossRef] [PubMed]
Garcia-Blanco MA Baraniak AP Lasda EL . Alternative splicing in disease and therapy. Nat Biotechnol. 2004;22:535–546. [CrossRef] [PubMed]
Rosenberg T Haim M Hauch AM Parving A . The prevalence of Usher syndrome and other retinal dystrophy-hearing impairment associations. Clin Genet. 1997;51:314–321. [CrossRef] [PubMed]
Fu XD Maniatis T . The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3′ splice site. Proc Natl Acad Sci U S A. 1992;89:1725–1729. [CrossRef] [PubMed]
D'Souza I Schellenberg GD . Determinants of 4-repeat tau expression. Coordination between enhancing and inhibitory splicing sequences for exon 10 inclusion. J Biol Chem. 2000;275:17700–17709. [CrossRef] [PubMed]
Ishii S Nakao S Minamikawa-Tachino R Desnick RJ Fan JQ . Alternative splicing in the alpha-galactosidase A gene: increased exon inclusion results in the Fabry cardiac phenotype. Am J Hum Genet. 2002;70:994–1002. [CrossRef] [PubMed]
Brunner S Colman D Travis AJ . Overexpression of RPGR leads to male infertility in mice due to defects in flagellar assembly. Biol Reprod. 2008 ;79 :608–617. [CrossRef] [PubMed]
Banin E Mizrahi-Meissonnier L Neis R . A non-ancestral RPGR missense mutation in families with either recessive or semi-dominant X-linked retinitis pigmentosa. Am J Med Genet A. 2007;143A:1150–1158. [CrossRef] [PubMed]
Walia S Fishman GA Swaroop A . Discordant phenotypes in fraternal twins having an identical mutation in exon ORF15 of the RPGR gene. Arch Ophthalmol. 2008;126:379–384. [CrossRef] [PubMed]
Figure 1.
 
Characterization of a splice site mutation in LCL 2616. RPGR transcript analysis in the patient cell line 2616 and the unaffected control using different primer combinations that span the whole RPGR transcript. DNA marker sizes are given in base pairs.
Figure 1.
 
Characterization of a splice site mutation in LCL 2616. RPGR transcript analysis in the patient cell line 2616 and the unaffected control using different primer combinations that span the whole RPGR transcript. DNA marker sizes are given in base pairs.
Figure 2.
 
Overexpression of the RPGR +Ex11a transcript isoform in patient cell line 4767. (A) RT-PCR amplification of RPGR transcripts containing exons 11 through 15. Schematic drawings illustrate the exon composition of amplified RT-PCR products. Primer binding sites are indicated as horizontal gray bars. DNA marker sizes are given in base pairs. (B) Sequence of the novel RPGR exon 11a and flanking intronic regions. Nucleotides in gray represent exon 11a sequences, whereas deduced amino acids are indicated as single letter code above of each nucleotide triplet. The stop codon is presented in italics, and coding sequences are shown in uppercase letters. The position mutated in patient 4767 (g.31259G>A) is marked by a box. NC_000023.9 was used as reference sequence for genomic DNA (numbers at the right indicate the nucleotide position). (C) Real-time quantitative RT-PCR of exon 11a expression levels detected in the unaffected control (I), in a pool of six LCLs carrying hemizygous RPGR mutations (n = 6) different from the sequence alteration in exon 11a (II) and in the patient cell line 4767 (III). Error bars represent 95% CI calculated from cDNA batches generated from the RNA of three independently grown LCL cells. Asterisk indicates statistical significance between measurements. (D) Electropherograms from parts of the exon 11a sequence of patient cell line 4767 and a control. Arrows indicate the position of the mutation g.31259G>A in patient 4767. The stop codon is shown in italics. Positions of putative binding sites for ESEs SF2/ASF and SC35 are depicted as horizontal bars. The results suggest the existence of an additional SC35 binding site uniquely in the patient.
Figure 2.
 
Overexpression of the RPGR +Ex11a transcript isoform in patient cell line 4767. (A) RT-PCR amplification of RPGR transcripts containing exons 11 through 15. Schematic drawings illustrate the exon composition of amplified RT-PCR products. Primer binding sites are indicated as horizontal gray bars. DNA marker sizes are given in base pairs. (B) Sequence of the novel RPGR exon 11a and flanking intronic regions. Nucleotides in gray represent exon 11a sequences, whereas deduced amino acids are indicated as single letter code above of each nucleotide triplet. The stop codon is presented in italics, and coding sequences are shown in uppercase letters. The position mutated in patient 4767 (g.31259G>A) is marked by a box. NC_000023.9 was used as reference sequence for genomic DNA (numbers at the right indicate the nucleotide position). (C) Real-time quantitative RT-PCR of exon 11a expression levels detected in the unaffected control (I), in a pool of six LCLs carrying hemizygous RPGR mutations (n = 6) different from the sequence alteration in exon 11a (II) and in the patient cell line 4767 (III). Error bars represent 95% CI calculated from cDNA batches generated from the RNA of three independently grown LCL cells. Asterisk indicates statistical significance between measurements. (D) Electropherograms from parts of the exon 11a sequence of patient cell line 4767 and a control. Arrows indicate the position of the mutation g.31259G>A in patient 4767. The stop codon is shown in italics. Positions of putative binding sites for ESEs SF2/ASF and SC35 are depicted as horizontal bars. The results suggest the existence of an additional SC35 binding site uniquely in the patient.
Figure 3.
 
Upregulation of the novel RPGR skipEx12 transcript. (A) RT-PCR amplification of exons 11 to 19 of the RPGR transcript. Schematic drawings illustrate the sequence of amplified RT-PCR products. Each RT-PCR product is marked by a number corresponding to the respective schematic drawing. Primer binding sites in exons 11 and 19 are indicated by horizontal gray bars. Numbers at the left represent DNA marker sizes and are given in base pairs. (B) RT-PCR of RPGR exons 11 to 19 of patient cell line 2550 and a control, either incubated with (+) or without (−) CHX to inhibit NMD. The RT-PCR reaction lacking a template is indicated as H2O. (C) Graphical illustration of mutation-induced mechanisms in patient cell line 2550 leading to detectable levels of RPGR skipEx12 transcripts. The asterisk labels a premature termination codon (PTC) in exon 12, which is generated in patient cell line 2550 because of the 4-bp deletion in exon 11. The PTC causes reduced expression levels of transcripts, including exon 12, by NMD (upper). In contrast, transcripts lacking exon 12 in combination with the 4-bp deletion escape NMD because of preservation of the open reading frame (lower).
Figure 3.
 
Upregulation of the novel RPGR skipEx12 transcript. (A) RT-PCR amplification of exons 11 to 19 of the RPGR transcript. Schematic drawings illustrate the sequence of amplified RT-PCR products. Each RT-PCR product is marked by a number corresponding to the respective schematic drawing. Primer binding sites in exons 11 and 19 are indicated by horizontal gray bars. Numbers at the left represent DNA marker sizes and are given in base pairs. (B) RT-PCR of RPGR exons 11 to 19 of patient cell line 2550 and a control, either incubated with (+) or without (−) CHX to inhibit NMD. The RT-PCR reaction lacking a template is indicated as H2O. (C) Graphical illustration of mutation-induced mechanisms in patient cell line 2550 leading to detectable levels of RPGR skipEx12 transcripts. The asterisk labels a premature termination codon (PTC) in exon 12, which is generated in patient cell line 2550 because of the 4-bp deletion in exon 11. The PTC causes reduced expression levels of transcripts, including exon 12, by NMD (upper). In contrast, transcripts lacking exon 12 in combination with the 4-bp deletion escape NMD because of preservation of the open reading frame (lower).
Figure 4.
 
Relative abundance of alternative transcript isoforms of RPGR in human retina. Expression levels of alternative variants are displayed as percentages of the constitutive RPGR transcript in human retina. Numbers in bars represent mean values given in percentages Error bars represent confidence intervals of 95% CIs calculated from three to four independently generated cDNA batches.
Figure 4.
 
Relative abundance of alternative transcript isoforms of RPGR in human retina. Expression levels of alternative variants are displayed as percentages of the constitutive RPGR transcript in human retina. Numbers in bars represent mean values given in percentages Error bars represent confidence intervals of 95% CIs calculated from three to four independently generated cDNA batches.
Figure 5.
 
Multi-tissue expression analyses of novel and previously reported RPGR transcript isoforms. Quantitative RT-PCR to detect expression of (A) RPGR +Ex11a, (B) RPGR skipEx12, (C) RPGR skipEx14, (D) RPGR skipEx15, (E) RPGR +Ex9a, and (F) RPGR skipEx14/15 from different donor tissues. For quantification of the novel RPGR transcript isoform, we used expression of the constitutive RPGR transcript as endogenous control. Subsequently, relative expression levels of each isoform were compared with human retina (retinal levels were set to 1). Asterisks illustrate significance between measurements as described in the Results section. Error bars represent 95% CIs calculated from three to four independently generated cDNA batches.
Figure 5.
 
Multi-tissue expression analyses of novel and previously reported RPGR transcript isoforms. Quantitative RT-PCR to detect expression of (A) RPGR +Ex11a, (B) RPGR skipEx12, (C) RPGR skipEx14, (D) RPGR skipEx15, (E) RPGR +Ex9a, and (F) RPGR skipEx14/15 from different donor tissues. For quantification of the novel RPGR transcript isoform, we used expression of the constitutive RPGR transcript as endogenous control. Subsequently, relative expression levels of each isoform were compared with human retina (retinal levels were set to 1). Asterisks illustrate significance between measurements as described in the Results section. Error bars represent 95% CIs calculated from three to four independently generated cDNA batches.
Table 1.
 
RPGR Mutations of Analyzed Lymphoblastoid Cell Lines Derived from Male Patients with X1RP
Table 1.
 
RPGR Mutations of Analyzed Lymphoblastoid Cell Lines Derived from Male Patients with X1RP
Patient ID Mutation in Exon/Intron Sequence Alteration Consequence of Sequence Alteration
2616 Intron 1 c.28+1G>A Potential splice defect
4767* Exon 5 c.389T>G p.Phe130Cys
Exon 11a g.31259G>A† ‡ Splice defect/p.Asp484Asn
2884 Exon 7 c.706C>T p.Glu236X
2555 Exon 8 c.823G>A p.Gly275Ser
2603 Exon 8 c.823G>A p.Gly275Ser
2549 Exon 11 c.1399C>T p.Gln467X
2550§ Exon 11 c.1402_1405delCCAG p.Arg468fs
Supplementary Table S1
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